Label free analysis of nucleic acids

ABSTRACT

A method is provided for label free analysis of nucleic acid materials. In accordance with the present invention, a fluorescent material is provided having a certain fluorescence emission without nucleic acids attached thereto. The fluorescent material of the present invention, has a greater emission upon the binding of a single stranded nucleic acid and an even greater emission upon the binding of a double stranded nucleic acid allowing detection of double stranded binding without using a label attached to the DNA.

FIELD OF THE INVENTION

The present invention relates to the detection of nucleic acids. Moreparticularly, the present invention relates to the detection of doublestranded nucleic acid hybridization without the use of any type of labelattached to the nucleic acid.

BACKGROUND OF THE INVENTION

Since virtually the inception of molecular biology, the detection ofnucleic acids (both DNA and RNA) has turned on the incorporation ofvarious labels into these molecules. At first, radioactive labels suchas ³²P were widely using in southerns, northerns and sequencing. Later,a great number of non-radioactive labels were developed, having groupswhich either fluoresced themselves or fluoresced upon interactions witha second molecule (e.g. biotin). It is an object of the presentinvention to develop methods and instruments which are capable ofdetecting nucleic acids without introduction of any type of exogenouslabel.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention, a method fordetecting label free analysis of nucleic acid binding is presented, themethod having the steps of providing a fluorescent polymeric matrixhaving a first fluorescent emission intensity; attaching a singlestranded DNA or RNA species to said fluorescent matrix to provide asecond, greater fluorescent intensity; and hybridizing said singlestranded RNA or DNA to a homologous nucleic acid, causing a furtherincrease in said fluorescent intensity, allowing detection of saidhybridization of said homologous nucleic acid binding.

DETAILED DESCRIPTION OF THE INVENTION A. GENERAL

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication No. WO 99/36760) and PCT/US01/04285(International Publication No. WO 01/58593), which are all incorporatedherein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos.10/442,021, 10/013,598 (U.S. patent application Publication20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodiedin U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, for example, PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,1594,965,188,and 5,333,675, and each of which is incorporated herein byreference in their entireties for all purposes. The sample may beamplified on the array. See, for example, U.S. Pat. No. 6,300,070 andU.S. Ser. No. 09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. No. 6,361,947, 6,391,592 and U.S.Ser. Nos. 09/916,135, 09/920,491 (U.S. patent application Publication20030096235), Ser. No. 09/910,292 (U.S. patent application Publication20030082543), and Ser. No. 10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol.152, Guide to Molecular Cloning Techniques (Academic Press, Inc., SanDiego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Ser. No. 10/389,194 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194,60/493,495 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S.Pat. No. 6,420,108.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (U.S.Publication No. 20020183936), Ser. Nos. 10/065,856, 10/065,868,10/328,818, 10/328,872, 10/423,403, and 60/482,389.

B. DEFINITIONS

The term “array” as used herein refers to an intentionally createdcollection of molecules which can be prepared either synthetically orbiosynthetically. The molecules in the array can be identical ordifferent from each other. The array can assume a variety of formats,for example, libraries of soluble molecules; libraries of compoundstethered to resin beads, silica chips, or other solid supports.

The term “monomer” as used herein refers to a single unit of polymer,which can be linked with the same or other monomers to form a biopolymer(for example, a single amino acid or nucleotide with two linking groupsone or both of which may have removable protecting groups) or a singleunit which is not part of a biopolymer. Thus, for example, a nucleotideis a monomer within an oligonucleotide polymer, and an amino acid is amonomer within a protein or peptide polymer; antibodies, antibodyfragments, chromosomes, plasmids, mRNA, cRNA, tRNA etc., for example,are also polymers.

The term “biopolymer” or sometimes refer by “biological polymer” as usedherein is intended to mean repeating units of biological or chemicalmoieties. Representative biopolymers include, but are not limited to,nucleic acids, oligonucleotides, amino acids, proteins, peptides,hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides,phospholipids, synthetic analogues of the foregoing, including, but notlimited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, andcombinations of the above. It is important to note that biopolymers andpolymers are not mutually exclusive. Proteins, enzymes, DNA,polyethylene, RNA, are all polymers as they are derived from a repeatingmonomer units. However, proteins, enzymes, DNA are all biopolymers asmany of them first appeared in nature. Sometimes, it is not easy toclassify something as a biopolymer or a polymer. For example, vastnumber of human made amino acid derivatives and nucleotide derivativeshave been created and polymerized. Some of these are based on naturalproducts, many more are not. At this point the distinction between thetwo can be somewhat semantical.

The term “biopolymer synthesis” as used herein is intended to encompassthe synthetic production, both in situ (in the cell) and synthetically,e.g. by organic synthetic techniques outside of the cell, of abiopolymer. Related to a bioploymer is a “biomonomer”.

The term “combinatorial synthesis strategy” as used herein refers to acombinatorial synthesis strategy is an ordered strategy for parallelsynthesis of diverse polymer sequences by sequential addition ofreagents which may be represented by a reactant matrix and a switchmatrix, the product of which is a product matrix. A reactant matrix is al column by m row matrix of the building blocks to be added. The switchmatrix is all or a subset of the binary numbers, preferably ordered,between I and m arranged in columns. A “binary strategy” is one in whichat least two successive steps illuminate a portion, often half, of aregion of interest on the substrate. In a binary synthesis strategy, allpossible compounds which can be formed from an ordered set of reactantsare formed. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme. A combinatorial “masking” strategy is a synthesis which useslight or other spatially selective deprotecting or activating agents toremove protecting groups from materials for addition of other materialssuch as amino acids.

The term “complementary” as used herein refers to the hybridization orbase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), or C and G. Two singlestranded RNA or DNA molecules are said to be complementary when thenucleotides of one strand, optimally aligned and compared and withappropriate nucleotide insertions or deletions, pair with at least about80% of the nucleotides of the other strand, usually at least about 90%to 95%, and more preferably from about 98 to 100%. Alternatively,complementarity exists when an RNA or DNA strand will hybridize underselective hybridization conditions to its complement. Typically,selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984),incorporated herein by reference.

The term “copolymer” refers to a polymer that is composed of more thanone monomer. Copolymers may be prepared by polymerizing one or moremonomers to provide a copolymer.

The term “detectable moiety” (Q) means a chemical group that provides asignal. The signal is detectable by any suitable means, includingspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. In certain cases, the signal is detectable by2 or more means.

The detectable moiety provides the signal either directly or indirectly.A direct signal is produced where the labeling group spontaneously emitsa signal, or generates a signal upon the introduction of a suitablestimulus. Radiolabels, such as ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P, and magneticparticles, such as Dynabeads™, are nonlimiting examples of groups thatdirectly and spontaneously provide a signal. Labeling groups thatdirectly provide a signal in the presence of a stimulus include thefollowing nonlimiting examples: colloidal gold (40-80 nm diameter),which scatters green light with high efficiency; fluorescent labels,such as fluorescein, Texas red, Rhoda mine, and green fluorescentprotein (Molecular Probes, Eugene, Oreg.), which absorb and subsequentlyemit light; chemiluminescent or bioluminescent labels, such as luminol,lophine, acridine salts and luciferins, which are electronically excitedas the result of a chemical or biological reaction and subsequently emitlight; spin labels, such as vanadium, copper, iron, manganese andnitroxide free radicals, which are detected by electron spin resonance(ESR) spectroscopy; dyes, such as quinoline dyes, triarylmethane dyesand acridine dyes, which absorb specific wavelengths of light; andcolored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)beads. See U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241.

A detectable moiety provides an indirect signal where it interacts witha second compound that spontaneously emits a signal, or generates asignal upon the introduction of a suitable stimulus. Biotin, forexample, produces a signal by forming a conjugate with streptavidin,which is then detected. See Hybridization With Nucleic Acid Probes. InLaboratory Techniques in Biochemistry and Molecular Biology; Tijssen,P., Ed.; Elsevier: New York, 1993; Vol. 24. An enzyme, such ashorseradish peroxidase or alkaline phosphatase, that is attached to anantibody in a label-antibody-antibody as in an ELISA assay, alsoproduces an indirect signal.

A preferred detectable moiety is a fluorescent group. Fluorescent groupstypically produce a high signal to noise ratio, thereby providingincreased resolution and sensitivity in a detection procedure.Preferably, the fluorescent group absorbs light with a wavelength aboveabout 300 nm, more preferably above about 350 nm, and most preferablyabove about 400 nm. The wavelength of the light emitted by thefluorescent group is preferably above about 310 nm, more preferablyabove about 360 nm, and most preferably above about 410 nm.

The fluorescent detectable moiety is selected from a variety ofstructural classes, including the following nonlimiting examples: 1- and2-aminonaphthalene, p,p′diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene,bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolyl phenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodaminedyes); cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes andfluorescent proteins (e.g., green fluorescent protein,phycobiliprotein).

A number of fluorescent compounds are suitable for incorporation intothe present invention. Nonlimiting examples of such compounds includethe following: dansyl chloride; fluoresceins, such as3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate;N-phenyl-1-amino-8-sulfonatonaphthalene;N-phenyl-2-amino-6-sulfonatonaphthanlene;4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonapththalene-6-sulfonate; N-phenyl,N-methyl 2-aminonaphthalene-6-sulfonate; ethidium bromide; stebrine;auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamin;N,N′-dioctadecyloxacarbocycanine; N,N′-dihexyl oxacarbocyanine;merocyanine, 4-(3′-pyrenyl)butryate; d-3-aminodesoxy-equilenin;12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2′-(vinylene-p-phenylene)bisbenzoxazole; p-bis[2-(4-methyl-5-phenyloxazolyl)]benzene; 6-dimethylamino-1,2-benzophenzin; retinol;bis(3′-aminopyridinium)-1,10-decandiyl diiodide; sulfonaphthylhydrazoneof hellibrienin; chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-[p-(2-benzimidazolyl)phenyl]maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadizole; merocyanine 540; resorufin; rosebengal and 2,4-diphenyl-3(2H)-furanone. Preferably, the fluorescentdetectable moiety is a fluorescein or rhodamine dye.

Another preferred detectable moiety is colloidal gold. The colloidalgold particle is typically 40 to 80 nm in diameter. The colloidal goldmay be attached to a labeling compound in a variety of ways. In oneembodiment, the linker moiety of the nucleic acid labeling compoundterminates in a thiol group (—SH), and the thiol group is directly boundto colloidal gold through a dative bond. See Mirkin et al. Nature 1996,382, 607-609. In another embodiment, it is attached indirectly, forinstance through the interaction between colloidal gold conjugates ofantibiotin and a biotinylated labeling compound. The detection of thegold labeled compound may be enhanced through the use of a silverenhancement method. See Danscher et al. J. Histotech 1993, 16, 201-207.

The term “effective amount” as used herein refers to an amountsufficient to induce a desired result.

Although generally used herein to define separate regions containingdiffering polymer sequences, the term “feature” generally refers to anyelement, e.g., region, structure or the like, on the surface of asubstrate. Typically, substrates to be scanned, will have small featuresizes, and consequently, high feature densities on substrate surfaces.For example, individual features will typically have at least one of alength or width dimension that is no greater than 100 microns, andpreferably, no greater than 50 microns, and more preferably no greaterthan about 20 microns. Thus, for embodiments employing substrates havinga plurality of polymer sequences on their surfaces, each differentpolymer sequence will typically be substantially contained within asingle feature.

The term “fragmentation” refers to the breaking of nucleic acidmolecules into smaller nucleic acid fragments. In certain embodiments,the size of the fragments generated during fragmentation can becontrolled such that the size of fragments is distributed about acertain predetermined nucleic acid length.

The term “genome” as used herein is all the genetic material in thechromosomes of an organism. DNA derived from the genetic material in thechromosomes of a particular organism is genomic DNA. A genomic libraryis a collection of clones made from a set of randomly generatedoverlapping DNA fragments representing the entire genome of an organism.

The term “hybridization” as used herein refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” The proportion of the population ofpolynucleotides that forms stable hybrids is referred to herein as the“degree of hybridization.” Hybridizations are usually performed understringent conditions, for example, at a salt concentration of no morethan 1 M and a temperature of at least 25° C. For example, conditions of5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probehybridizations. For stringent conditions, see, for example, Sambrook,Fritsche and Maniatis. “Molecular Cloning A laboratory Manual” b 2 ^(nd)Ed. Cold Spring Harbor Press (1989) which is hereby incorporated byreference in its entirety for all purposes above.

The term “hybridization conditions” as used herein will typicallyinclude salt concentrations of less than about 1M, more usually lessthan about 500 mM and preferably less than about 200 mM. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and preferably in excessof about 37° C. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, the combination of parameters is more important thanthe absolute measure of any one alone.

The term “hybridization probes” as used herein are oligonucleotidescapable of binding in a base-specific manner to a complementary strandof nucleic acid. Such probes include peptide nucleic acids, as describedin Nielsen et al., Science 254, 1497-1500 (1991), and other nucleic acidanalogs and nucleic acid mimetics.

The term “hybridizing specifically to” as used herein refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (for example, total cellular)DNA or RNA.

The term “initiation monomer” or “initiator monomer” as used herein ismeant to indicate the first monomer which is covalently attached viareactive groups, e.g., nucleophiles and electrophiles to the surface ofthe polymer, or the first monomer which is attached to a linker orspacer arm attached to the polymer, the linker or spacer arm beingattached to the polymer via reactive groups.

The term “isolated nucleic acid” as used herein mean an object speciesinvention that is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition). Preferably, an isolated nucleic acid comprises at leastabout 50, 80 or 90% (on a molar basis) of all macromolecular speciespresent. Most preferably, the object species is purified to essentialhomogeneity (contaminant species cannot be detected in the compositionby conventional detection methods).

The term “ligand” as used herein refers to a molecule that is recognizedby a particular receptor. The agent bound by or reacting with a receptoris called a “ligand,” a term which is definitionally meaningful only interms of its counterpart receptor. The term “ligand” does not imply anyparticular molecular size or other structural or compositional featureother than that the substance in question is capable of binding orotherwise interacting with the receptor. Also, a ligand may serve eitheras the natural ligand to which the receptor binds, or as a functionalanalogue that may act as an agonist or antagonist. Examples of ligandsthat can be investigated by this invention include, but are notrestricted to, agonists and antagonists for cell membrane receptors,toxins and venoms, viral epitopes, hormones (for example, opiates,steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, substrate analogs, transition state analogs, cofactors,drugs, proteins, and antibodies.

The term “linkage disequilibrium” or sometimes refer by allelicassociation as used herein refers to the preferential association of aparticular allele or genetic marker with a specific allele, or geneticmarker at a nearby chromosomal location more frequently than expected bychance for any particular allele frequency in the population. Forexample, if locus X has alleles a and b, which occur equally frequently,and linked locus Y has alleles c and d, which occur equally frequently,one would expect the combination ac to occur with a frequency of 0.25.If ac occurs more frequently, then alleles a and c are in linkagedisequilibrium. Linkage disequilibrium may result from natural selectionof certain combination of alleles or because an allele has beenintroduced into a population too recently to have reached equilibriumwith linked alleles.

The term “mixed population” or sometimes refer by “complex population”as used herein refers to any sample containing both desired andundesired nucleic acids. As a non-limiting example, a complex populationof nucleic acids may be total genomic DNA, total genomic RNA or acombination thereof. Moreover, a complex population of nucleic acids mayhave been enriched for a given population but include other undesirablepopulations. For example, a complex population of nucleic acids may be asample which has been enriched for desired messenger RNA (mRNA)sequences but still includes some undesired ribosomal RNA sequences(rRNA).

The term “monomer” as used herein refers to any member of the set ofmolecules that can be joined together to form an oligomer or polymer.The set of monomers useful in the present invention includes, but is notrestricted to, for the example of (poly)peptide synthesis, the set ofL-amino acids, D-amino acids, or synthetic amino acids. As used herein,“monomer” refers to any member of a basis set for synthesis of anoligomer. For example, dimers of L-amino acids form a basis set of 400“monomers” for synthesis of polypeptides. Different basis sets ofmonomers may be used at successive steps in the synthesis of a polymer.The term “monomer” also refers to a chemical subunit that can becombined with a different chemical subunit to form a compound largerthan either subunit alone.

The term “mRNA,” or sometimes referred to as “mRNA transcripts,” as usedherein, includes, but not limited to pre-mRNA transcript(s), transcriptprocessing intermediates, mature mRNA(s) ready for translation andtranscripts of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). Transcript processing may include splicing, editing anddegradation. As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed fromthat cDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, mRNA derivedsamples include, but are not limited to, mRNA transcripts of the gene orgenes, cDNA reverse transcribed from the mRNA, cRNA transcribed from thecDNA, DNA amplified from the genes, RNA transcribed from amplified DNA,and the like.

The term “nucleic acid library” or sometimes refer by “array” as usedherein refers to an intentionally created collection of nucleic acidswhich can be prepared either synthetically or biosynthetically andscreened for biological activity in a variety of different formats (forexample, libraries of soluble molecules; and libraries of oligostethered to resin beads, silica chips, or other solid supports).Additionally, the term “array” is meant to include those libraries ofnucleic acids which can be prepared by spotting nucleic acids ofessentially any length (for example, from 1 to about 1000 nucleotidemonomers in length) onto a substrate. The term “nucleic acid” as usedherein refers to a polymeric form of nucleotides of any length, eitherribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs),that comprise purine and pyrimidine bases, or other natural, chemicallyor biochemically modified, non-natural, or derivatized nucleotide bases.The backbone of the polynucleotide can comprise sugars and phosphategroups, as may typically be found in RNA or DNA, or modified orsubstituted sugar or phosphate groups. A polynucleotide may comprisemodified nucleotides, such as methylated nucleotides and nucleotideanalogs. The sequence of nucleotides may be interrupted bynon-nucleotide components. Thus the terms nucleoside, nucleotide,deoxynucleoside and deoxynucleotide generally include analogs such asthose described herein. These analogs are those molecules having somestructural features in common with a naturally occurring nucleoside ornucleotide such that when incorporated into a nucleic acid oroligonucleoside sequence, they allow hybridization with a naturallyoccurring nucleic acid sequence in solution. Typically, these analogsare derived from naturally occurring nucleosides and nucleotides byreplacing and/or modifying the base, the ribose or the phosphodiestermoiety. The changes can be tailor made to stabilize or destabilizehybrid formation or enhance the specificity of hybridization with acomplementary nucleic acid sequence as desired.

The term “nucleic acids” as used herein may include any polymer oroligomer of pyrimidine and purine bases, preferably cytosine, thymine,and uracil, and adenine and guanine, respectively. See Albert L.Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982).Indeed, the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligomers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally-occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

The term “PVP” refers to polyvinylpyrrolidone, which has the structure:

The term “oligonucleotide” or sometimes refer by “polynucleotide” asused herein refers to a nucleic acid ranging from at least 2, preferableat least 8, and more preferably at least 20 nucleotides in length or acompound that specifically hybridizes to a polynucleotide.Polynucleotides of the present invention include sequences ofdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may beisolated from natural sources, recombinantly produced or artificiallysynthesized and mimetics thereof. A further example of a polynucleotideof the present invention may be peptide nucleic acid (PNA). Theinvention also encompasses situations in which there is a nontraditionalbase pairing such as Hoogsteen base pairing which has been identified incertain tRNA molecules and postulated to exist in a triple helix.“Polynucleotide” and “oligonucleotide” are used interchangeably in thisapplication.

The term “polymorphism” as used herein refers to the occurrence of twoor more genetically determined alternative sequences or alleles in apopulation. A polymorphic marker or site is the locus at whichdivergence occurs. Preferred markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allelic form is arbitrarily designated as the reference formand other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wildtype form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Adiallelic polymorphism has two forms. A triallelic polymorphism hasthree forms. Single nucleotide polymorphisms (SNPs) are included inpolymorphisms.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions for example,buffer and temperature, in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, for example, DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from 15 to 30 nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template. A primer need not reflect the exactsequence of the template but must be sufficiently complementary tohybridize with such template. The primer site is the area of thetemplate to which a primer hybridizes. The primer pair is a set ofprimers including a 5′ upstream primer that hybridizes with the 5′ endof the sequence to be amplified and a 3′ downstream primer thathybridizes with the complement of the 3′ end of the sequence to beamplified.

The term “probe” as used herein refers to a surface-immobilized moleculethat can be recognized by a particular target. See U.S. Pat. No.6,582,908 for an example of arrays having all possible combinations ofprobes with 10, 12, and more bases. Examples of probes that can beinvestigated by this invention include, but are not restricted to,agonists and antagonists for cell membrane receptors, toxins and venoms,viral epitopes, hormones (for example, opioid peptides, steroids, etc.),hormone receptors, peptides, enzymes, enzyme substrates, cofactors,drugs, lectins, sugars, oligonucleotides, nucleic acids,oligosaccharides, proteins, and monoclonal antibodies.

The term “receptor” as used herein refers to a molecule that has anaffinity for a given ligand. Receptors may be naturally-occurring ormanmade molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Receptors may be attached,covalently or noncovalently, to a binding member, either directly or viaa specific binding substance. Examples of receptors which can beemployed by this invention include, but are not restricted to,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses, cellsor other materials), drugs, polynucleotides, nucleic acids, peptides,cofactors, lectins, sugars, polysaccharides, cells, cellular membranes,and organelles. Receptors are sometimes referred to in the art asanti-ligands. As the term receptors is used herein, no difference inmeaning is intended. A “Ligand Receptor Pair” is formed when twomacromolecules have combined through molecular recognition to form acomplex. Other examples of receptors which can be investigated by thisinvention include but are not restricted to those molecules shown inU.S. Pat. No. 5,143,854, which is hereby incorporated by reference inits entirety.

The term “solid support”, “support”, and “substrate” as used herein areused interchangeably and refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In many embodiments,at least one surface of the solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. See U.S. Pat. No.5,744,305 for exemplary substrates.

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally-occurring orman-made molecules. Also, they can be employed in their unaltered stateor as aggregates with other species. Targets may be attached, covalentlyor noncovalently, to a binding member, either directly or via a specificbinding substance. Examples of targets which can be employed by thisinvention include, but are not restricted to, antibodies, cell membranereceptors, monoclonal antibodies and antisera reactive with specificantigenic determinants (such as on viruses, cells or other materials),drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins,sugars, polysaccharides, cells, cellular membranes, and organelles.Targets are sometimes referred to in the art as anti-probes. As the termtargets is used herein, no difference in meaning is intended. A “ProbeTarget Pair” is formed when two macromolecules have combined throughmolecular recognition to form a complex.

C. PLATFORM FOR LABEL FREE ANALYSIS OF GENOMIC DNA

In accordance with an aspect of the present invention, the platform forlabel free analysis of genomic DNA analysis relates generally todiagnostic and genomic analysis. An aspect of the present inventioncontemplates the use of a polymeric matrix and its use as a platform forlabel free analysis of genomic material. As is known to those of skillin the a great deal of time and effort has gone into the preparation,standardization, etc. for labels for the detection of genomic DNA andmRNA. While many successful labels have been created, it is not withoutcost. Indeed, labeling technology has become a significant amount of theexpense of running a nucleic acid array analysis.

The present invention relates to the use of polymeric matrices and theiruse as a platform for label free analysis of genomic material. Thesematrices comprise a fluorescent substrate that can be used as a supportfor DNA synthesis. This DNA/RNA layer has the effect of changing theemission intensity of the fluorescent substrate. Hybridization of theDNA/RNA material will cause a further change in emission intensity, andtherefore a recordable signal to be used for the genomic analysis(Scheme 1).

In accordance with an aspect of the present invention, it is postulatedthat the mechanism of change in emission intensity I upon hybridizationis due to the change in refractive index of the medium (η) experiencedby the emitted light before reaching the detector. The emissionintensity is proportional to the optical density at the excitationwavelength (OD), the quantum efficiency of the emitting species (Φ) andinversely proportional to the square of the refractive index of themedium (η), equation 1. $I = \frac{{OD}\quad\Phi}{\eta^{2}}$

Equation I. Relationship between emission intensity and refractiveindex.

The DNA/RNA layer will result into a change of the refractive index andtherefore into a change in the emission intensity (I), Scheme 2.

In accordance with an aspect of the present invention, several matricesmay be useful to this purpose. In general the matrices could be eitherfluorescent of made fluorescent by doping or covalently attaching afluorescent substrate.

EXAMPLE

As a preliminary result a fluorescent film(PVP-co-poly(N-2-methacryloxyethyl)methacrylamide attached to a3-acryloxypropryl trimethoxysilane (or a3-acrylamidopropyltrimethoxysilane) coated glass surface by spin-coatingand phtopolymerizing at 254 nm) was used as a matrix for the label freeanalysis of genomic DNA. The film itself had a fluorescence of I=4,000counts. When a layer of DNA was generated onto the surface, usingMenpoc-amidites, the fluorescence intensity increased to 8,000 counts.When a layer of DNA was generated onto the polymer, but with TCAchemistry, a more efficient chemistry, a larger signal was recordedI=12,000 counts. Finally the probe was exposed to the specific unlabeledtarget and let to hybridize at room temperature for, it resulted in afurther increase of the emission intensity (I=15,000 counts after only 1hour, I=20,000 counts after 17 hours.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many variations of the invention willbe apparent to those of skill in the art upon reviewing the abovedescription. All cited references, including patent and non-patentliterature, are incorporated herein by reference in their entireties forall purposes.

1. A method for detecting label free analysis of nucleic acid binding,said method comprising the steps of providing a fluorescent polymericmatrix having a first fluorescent emission intensity; attaching a singlestranded DNA or RNA species to said fluorescent matrix to provide asecond, greater fluorescent intensity; and hybridizing said singlestranded RNA or DNA to a homologous nucleic acid, causing a furtherincrease in said fluorescent intensity, allowing detection of saidhybridization of said homologous nucleic acids.